Propofol Alleviates Anxiety‐Like Behaviors Associated with Pain by Inhibiting the Hyperactivity of PVNCRH Neurons via GABAA Receptor β3 Subunits

Abstract Pain, a comorbidity of anxiety disorders, causes substantial clinical, social, and economic burdens. Emerging evidence suggests that propofol, the most commonly used general anesthetic, may regulate psychological disorders; however, its role in pain‐associated anxiety is not yet described. This study investigates the therapeutic potential of a single dose of propofol (100 mg kg−1) in alleviating pain‐associated anxiety and examines the underlying neural mechanisms. In acute and chronic pain models, propofol decreased anxiety‐like behaviors in the elevated plus maze (EPM) and open field (OF) tests. Propofol also reduced the serum levels of stress‐related hormones including corticosterone, corticotropin‐releasing hormone (CRH), and norepinephrine. Fiber photometry recordings indicated that the calcium signaling activity of CRH neurons in the paraventricular nucleus (PVNCRH) is reduced after propofol treatment. Interestingly, artificially activating PVNCRH neurons through chemogenetics interfered with the anxiety‐reducing effects of propofol. Electrophysiological recordings indicated that propofol decreases the activity of PVNCRH neurons by increasing spontaneous inhibitory postsynaptic currents (sIPSCs). Further, reducing the levels of γ‐aminobutyric acid type A receptor β3 (GABAA β3) subunits in PVNCRH neurons diminished the anxiety‐relieving effects of propofol. In conclusion, this study provides a mechanistic and preclinical rationale to treat pain‐associated anxiety‐like behaviors using a single dose of propofol.


Introduction
Pain, especially chronic pain, frequently leads to maladaptive psychological responses, including anxiety. [1]In particular, anxiety can exacerbate pain intensity and duration, potentially leading to DOI: 10.1002/advs.202309059 a vicious cycle of pain and anxiety, resulting in a considerable burden on patients, families, and society. [2]However, in a clinical setting, the management of physical pain is the primary focus, while the treatment of pain-associated emotional reactions, such as anxiety associated with pain, is often neglected.Effective medications and interventions to treat anxiety associated with pain could reduce the incidence of subsequent severe emotional disorders and improve patients quality of life.
Propofol, a drug that won the Lasker-DeBakey Clinical Medical Research Award, exerts general anesthetic effects via the -aminobutyric acid type A receptor (GABA A ). [3] In clinical practice, patients receiving propofol anesthesia commonly experience intense and enduring sensations of euphoria. [4]This implies that propofol is a promising drug for the treatment of mood disorders.4a] Additionally, clinical studies have suggested that propofol can alleviate perioperative anxiety by inhibiting the activation of the hypothalamicpituitary-adrenal (HPA) axis to maintain the homeostasis. [5]However, whether propofol can alleviate painassociated anxiety responses has not yet been determined, and its neural mechanisms and molecular targets have not yet been investigated.
Corticotropin-releasing hormone (CRH) neurons located in the paraventricular nucleus (PVN) of the hypothalamus are crucial for controlling the HPA stress-response axis. [6]Stress activates the PVN CRH neurons to release CRH.Subsequently, CRH enters the pituitary gland and initiates a cascading response, ultimately leading to the release of a large amount of corticosterone (CORT), which disrupts homeostasis and can eventually lead to mental health issues. [7]The activation of PVN CRH neurons is associated with an increased risk of anxiety.We have previously indicated that stress can induce increased excitability of PVN CRH neurons by disrupting synaptic homeostasis, resulting in anxiety-like behaviors and a surge in stress-related hormones in mice. [8]Moreover, chemogenetic activation of PVN CRH neurons promotes anxiety-like behaviors.Pain induces increased c-Fos expression in PVN CRH neurons, providing evidence that PVN CRH neurons rapidly respond to pain stimulation. [9]Furthermore, a pilot in vitro electrophysiological investigation has indicated that propofol acts on GABA A receptors in neurons within the PVN, resulting in enhanced inhibitory currents. [10]Based on these studies, PVN CRH neurons are likely to be crucial neural targets through which propofol may alleviate anxiety-like behaviors associated with pain.
In this study, we investigated whether the acute administration of different doses of propofol could modulate the excitability of PVN CRH neurons to regulate anxiety-like behaviors associated with pain.Using acute and chronic pain models, we demonstrated that a single dose of propofol (100 mg kg −1 ) alleviated anxiety-like behaviors and suppressed stress-related hormone surges.Furthermore, our findings demonstrate that propofol effectively reduces the calcium signaling activity in PVN CRH neurons.Local injection of propofol has been shown to alleviate anxiety-like behaviors in mice with CFA-induced pain.However, when we chemogenetically activated PVN CRH neurons blocked the anxiety-relieving effects of propofol.Additionally, we observed that the knockdown of -aminobutyric acid type A receptor 3 (GABA A 3) subunits in PVN CRH neurons attenuated the therapeutic effects of propofol.These results establish a foundation for the potential use of propofol to treat anxiety-like behaviors associated with pain.

Propofol Alleviates Anxiety-Like Behaviors and Endocrine Responses Associated with Pain
To examine the therapeutic effects of propofol on acute painassociated anxiety, we injected 25 μL of CFA into the left hind paw of mice to establish a model of inflammatory pain (Figure 1A). [11]our hours after the CFA injection, the mice displayed a significant reduction in their mechanical thresholds, as determined by von Frey tests (Figure 1B), and increased anxiety-like behaviors (Figure 1C-E).Specifically, the CFA mice spent 70% less time in the open arms of the elevated plus maze (EPM) test (Figure 1C) and 50% less time in the central zone of the open field (OF) test (Figure 1D) compared to the saline-treated mice; nevertheless, the total distance traveled did not change during the OF test between the CFA and the saline groups (Figure 1E).Additionally, time-course experiments indicated that anxiety-like behaviors and mechanical hyperalgesia persisted for up to 3 days after the CFA injection (Figure 1B; Figure S1, Supporting Information).These data, consistent with those of previous studies, confirm that pain induces anxiety-like behaviors in mice. [12]4a] Therefore, we were interested in whether a single dose of propofol (25, 50, or 100 mg kg −1 ) could reduce acute pain-associated anxiety-like behaviors.A single propofol dose of 100 mg kg −1 (but not 25 or 50 mg kg −1 ) administered to mice 10 min after the CFA injection alleviated anxiety-like behaviors 4 h after treatment (Figure 1G-I; Figure S2, Supporting Information), although 100 mg kg −1 propofol did not affect mechanical hypersensitivity (Figure 1F).We recently demonstrated that propofol has long-lasting anti-anhedonic effects in mice.Therefore, we implemented the EPM, OF, and von Frey tests 24 h and 3 days after a single dose of propofol (100 mg kg −1 ).Strikingly, the therapeutic effects of propofol on acute pain-associated anxiety-like behaviors were observed for at least 3 days after treatment (Figure S3, Supporting Information).Furthermore, we ruled out the effects of the solvent intralipid (Figure S4, Supporting Information).
Furthermore, we investigated stress-related hormones in the plasma of control, CFA+saline, and CFA+propofol mice.The CFA+saline mice exhibited increased levels of CORT, CRH, and norepinephrine (NE) compared to the control mice (Figure 1J).However, a single dose of propofol (100 mg kg −1 ) resulted in a significant decrease in these stress-related hormones (Figure 1J), and this inhibitory effect lasted for at least 3 days (Figure S5, Supporting Information).Thus, at both the behavioral and hormonal levels, a single dose of propofol (100 mg kg −1 ) produces rapid and long-lasting effects on anxiety-like behaviors associated with acute pain in mice.
Next, we conducted similar experiments to investigate the effects of propofol on anxiety-like behaviors and mechanical thresholds in the context of chronic pain (Figure 1K).First, we established a spared nerve injury (SNI) chronic pain model.Twenty-one days after the SNI surgery, the mice displayed a significant reduction in their mechanical thresholds for allodynia (Figure 1L) and anxiety-like behaviors (Figure 1M-O).Subsequently, we injected 100 mg kg −1 propofol to assess changes in anxiety-like behaviors and mechanical thresholds in the SNI mice.Similar to the results observed in the CFA-injected mice, propofol did not improve mechanical hyperalgesia in the SNI mice (Figure 1P).Behavioral results suggested that propofol alleviated anxiety-like behaviors associated with chronic pain (Figure 1Q-S), and this therapeutic effect was observed at least 3 days after treatment (Figure S6, Supporting Information).Finally, we established that propofol effectively inhibited the increase in the serum levels of CORT, CRH, and NE induced by chronic pain in the SNI mice, and this effect lasted for at least 3 days after treatment (Figure 1T; Figure S7, Supporting Information).
Overall, our results suggest that propofol alleviates anxietylike behaviors and endocrine responses associated with acute and chronic pain without improving mechanical hyperalgesia.

Propofol Reduces Pain-Induced Hyperactivity of PVN CRH Neurons in Mice
PVN CRH neurons trigger hormonal cascades along the HPA axis and coordinate stress-related behaviors. [6,7,13]To quantify the physiological activity of PVN CRH neurons during pain and propofol administration, we used fiber photometry to monitor calcium signal alterations in real-time in PVN CRH neurons of CRH-ires-Cre mice that had received an injection of the rAAV-hSyn-DIO-GCaMP7b vector in the PVN (Figure 2A,B).
First, we examined the effect of CFA-induced pain on calcium signaling in PVN CRH neurons, which exhibited increased PVN CRH neuronal activity following the CFA injection (Figure 2C,D).Subsequently, we measured the response dynamics of the PVN CRH neurons to different doses of propofol (25, 50, and 100 mg kg −1 ) in naïve CRH-ires-Cre mice.A single dose of 100 mg kg −1 propofol suppressed the calcium signals of PVN CRH neurons, and this effect persisted for up to 80-90 min after drug administration (Figure 2E,F).At doses of 25 and 50 mg kg −1 , propofol inhibited the activity of PVN neurons, and this inhibitory effect lasted for ≈40-60 min (Figure S8, Supporting Information).To determine the effect of propofol on pain-induced activation of PVN CRH neurons, mice were administered propofol 10 min after the CFA injection.The results indicated that propofol promptly reversed the neuronal activation induced by the CFA injection (Figure 2G,H).Furthermore, we recorded the effects of propofol on calcium activity in PVN CRH neurons in the SNI mouse model of chronic pain (Figure 2I).We established that propofol (100 mg kg −1 ) decreased PVN CRH neuronal activity in the SNI mice (Figure 2J,K).
Consistent with fiber photometry recordings, in vitro electrophysiological recordings revealed that propofol inhibited the increase in pain-induced action potentials (APs).We recorded the activity of CRH neurons in PVN slices from the control, CFA+saline, and CFA+propofol mice.APs were evoked by 25-pA step depolarizing current pulses ranging from −25 to 200 pA.In the CFA+saline mice, the PVN CRH neurons exhibited increased neuronal excitability, which was reversed by propofol treatment (Figure 2L,M).Additionally, we recorded the activity of CRH neurons in PVN slices from the sham, SNI+saline, and SNI+propofol mice.PVN CRH neurons in the SNI+saline mice also exhibited increased excitability compared to sham mice, whereas propofol inhibited neuronal firing (Figure 2N,O).
These results indicate that propofol decreases the hyperactivation of PVN CRH neurons induced by pain.

Inhibiting PVN CRH Neurons Alleviates Pain-Associated Anxiety-Like Behaviors
Given the hyperactivity of PVN CRH neurons in response to anxiety-like behaviors induced by pain, we assessed whether inhibiting PVN CRH neurons could reverse anxiety-like behaviors caused by pain.We administered rAAV-hSyn-DIO-hM4D(Gi)-mCherry (hM4D(Gi) group) or rAAV-hSyn-DIO-mCherry (mCherry group) bilaterally into the PVN of CFA mice (Figure 3A).The perfusion of brain slices with clozapine N-oxide (CNO) decreased the firing rate of PVN CRH neurons (Figure S9, Supporting Information).Compared to mice in the mCherry group, mice in the hM4D(Gi) group spent more time in the open arms of the EPM test (Figure 3B) and in the central zone of the OF test (Figure 3C), without exhibiting any effects on the total distance traveled (Figure 3D) or paw withdrawal threshold (Figure 3E), implying that chemogenetic inhibition of PVN CRH neurons rescued anxiety-like behaviors in the CFA-4h mice.Similar results were observed in the SNI mice (Figure 3F-J).

Propofol Rapidly Attenuates Anxiety-Like Behaviors Through the Inhibition of PVN CRH Neurons
To further establish the causal role of PVN CRH neurons in the ability of propofol to alleviate anxiety-like behaviors, we first conducted an experiment in which propofol was locally injected into the PVN of mice subjected to CFA.We established that the local administration of propofol to the brain significantly mitigated pain-associated anxiety-like behaviors (Figure S10, Supporting Information).Subsequently, we injected rAAV-hSyn-DIO-hM3D(Gq)-mCherry and rAAV-hSyn-DIO-mCherry bilaterally into the PVN of saline-and CFA mice (Figure 4A,B).The EPM test for CFA + saline and CFA + propofol (100 mg kg −1 ) groups at 4 h after drug injection (n = eight mice per group).H) Time in the central zone during the OF test for CFA + saline and CFA + propofol (100 mg kg −1 ) groups at 4 h after drug injection (n = twelve mice per group).I) Total distance traveled during the OF test in CFA + saline and CFA + propofol (100 mg kg −1 ) groups at 4 h after drug injection (n = twelve mice per group).J) Serum CORT concentrations (left), serum CRH concentrations (middle), and serum NE concentrations (right) for control, CFA + saline, and CFA + propofol (100 mg kg −1 ) groups at 4 h after drug injection (n = five mice per group).K) Schematic of the surgery of SNI and propofol along with behavioral and stress-related hormones tests.L) Paw withdrawal threshold over time for sham and SNI groups (n = eight mice per group) M) Time in the open arms during the EPM test for sham and SNI groups at 21 days after surgery (n = seven mice per group).N) Time in the central zone during the OF test for sham and SNI groups at 21 days after surgery (n = seven mice per group).O) Total distance traveled during the OF test for sham and SNI groups at 21 days after surgery (n = seven mice per group).P) Paw withdrawal threshold over time for SNI + saline and SNI + propofol (100 mg kg −1 ) groups (n = eight mice per group).Q) Time in the open arms during the EPM test for SNI + saline and SNI + propofol (100 mg kg −1 ) groups at 4 h after drug injection (n = seven mice per group).R) Time in the central zone during the OF test for SNI + saline and SNI + propofol (100 mg kg −1 ) groups at 4 h after drug injection (n = seven mice per group).S) Total distance traveled during the OF test in SNI + saline and SNI + propofol (100 mg kg −1 ) groups at 4 h after drug injection (n = seven mice per group).T) Serum CORT concentrations (left), serum CRH concentrations (middle), and serum NE concentrations (right) for sham, SNI + saline, and SNI + propofol (100 mg kg −1 ) groups at 4 h after drug injection (n = five mice per group).Data are shown as the mean (white circles) ± SD (vertical lines) along with individual data points and were compared using two-tailed, unpaired Student's t-test (C-E, G-I, M-O and Q-S) or two-way ANOVA followed by Tukey's multiple comparisons test (B, F, L and P) or one-way ANOVA followed by Tukey's multiple comparisons test (J and T).CFA, complete Freund's adjuvant; SNI, spared nerve injury; EPM, elevated plus maze; OF, open field; ELISA, enzyme linked immunosorbent assay; CORT, corticosterone; CRH, corticotropin-releasing hormone; NE, norepinephrine; BL, baseline; CFA + Saline, CFA-injected mice exposed to saline; CFA + Propofol, CFA-injected mice exposed to propofol; SNI + Saline, SNI mice exposed to saline; SNI + Propofol, SNI mice exposed to propofol.perfusion of brain slices with CNO increased the firing rate of PVN CRH neurons (Figure S9, Supporting Information).Chemogenetic activation of PVN CRH neurons blocked the anxietyrelieving effects of propofol in the CFA mice (Figure 4C-E).Similar results were observed in the SNI mice (Figure 4F-J).In addition to our earlier findings, these results suggest that PVN CRH neurons may be the neural target of propofol in anxiety-like behaviors associated with pain.

Propofol Rebalances the Synaptic Homeostasis in PVN CRH Neurons Through Increased Spontaneous Inhibitory Postsynaptic Currents
To further elucidate the changes in intrinsic excitability in PVN CRH neurons triggered by pain and propofol administration, we used whole-cell patch-clamp recordings to study how propofol affects PVN CRH neurons at the cellular level (Figure 5A,H).
To examine the synaptic mechanism underlying the effects of propofol in alleviating anxiety-like behaviors during pain, we recorded spontaneous inhibitory postsynaptic currents (sIP-SCs) and spontaneous excitatory postsynaptic currents (sEP-SCs) in PVN CRH neurons.The frequency and amplitude of the sIPSCs decreased in the CFA+saline group 4 h after treatment (Figure 5B-D).The amplitude of the sIPSCs in the CFA+propofol group was significantly higher than that in the CFA+saline group at this time point; however, their frequencies did not differ (Figure 5B-D).The frequency of sEPSCs increased in the CFA+saline group 4 h after treatment (Figure 5E,F), whereas the amplitude of sEPSCs did not differ between the control and CFA+saline groups (Figure 5E,G), and the frequency and amplitude of sEPSCs did not differ between the CFA+propofol and CFA+saline groups (Figure 5E-G).
Additionally, SNI induced a notable decrease in the amplitude and frequency of sIPSCs (Figure 5I-K), whereas sEPSCs remained unchanged, indicating weakened inhibitory synaptic transmission of PVN CRH neurons (Figure 5L-N).Propofol increased the frequency and amplitude of sIPSCs (Figure 5I-K), but no alteration was observed in sEPSCs 4 h after treatment (Figure 5L-N).
Overall, these data demonstrate that propofol administration rebalances synaptic homeostasis in PVN CRH neurons by increasing sIPSCs in mouse pain models.

Propofol Attenuates Anxiety-Like Behaviors Associated with Pain Through GABA A 𝜷3 Subunits in PVN CRH Neurons
The general anesthetic effects of propofol are exerted primarily through its interaction with GABA A . [3]Propofol can activate GABA A directly or enhance its response to GABA when applied along with this neurotransmitter or other agonists. [14]The 3 subunit of GABA A is one of the important targets of propofol. [15]urthermore, pain can decrease expression of the 3 subunits. [16]o explore the molecular targets of propofol's anxiety-relieving effects, we performed western blotting experiments to determine the expression level of GABA A 3 subunits in the PVN.The CFA injection resulted in a decrease in the expression level of the GABA A 3 subunits in the PVN, which was reversed in the propofol-treated group (Figure 6A,B).
Subsequently, we performed a bilateral injection of the rAAV-DIO-shRNA (Gabrb3)-GABA A 3 vector into the PVN of CRHires-Cre mice to knock down the GABA A 3 expression specifically in PVN CRH neurons (Figure 6C,D).The downregulation of GABA A 3 subunits expression in PVN CRH neurons reduced the anxiety-relieving effects of propofol in the CFA mice at 4 h (Figure 6E-G), 24 h, and 3 days after treatment (Figure S11, Supporting Information) as well as in the SNI mice at 4 h (Figure 6H-L), 24 h, and 3 days after treatment (Figure S12, Supporting Information).Notably, knocking down the GABA A 3 subunits expression in PVN CRH neurons did not alter mechanical hyperalgesia in naïve mice, which provides evidence that propofol has no analgesic effect in this system (Figure S13, Supporting Information).These findings suggest that the GABA A 3 subunits are likely involved in the mechanism by which propofol provides long-term reduction in anxiety-like behaviors associated with pain.

Discussion
This study demonstrated that a single dose of propofol (100 mg kg −1 ) alleviates the anxiety-like behaviors associated with pain.This therapeutic effect is achieved by acting on GABA A 3 subunits, which restores excitation-inhibition (E-I) balance and subsequently inhibits the excessive activation of PVN CRH neurons in mouse pain models (Figure 7).These results provide an experimental basis for novel clinical applications of propofol and support the feasibility of targeted treatment of emotional components of pain.

Efficacy of Propofol as a Pharmacological Intervention for Pain-Related Anxiety
Owing to its ability to induce euphoric moods and provide lasting relaxation, propofol has the potential to be a treatment option for mood disorders. [4]We used models of acute and chronic pain to evaluate the effects of propofol on anxiety-like behaviors associated with pain.Propofol rapidly improves anxiety-like behaviors associated with acute and chronic pain, and this anxiolytic effect lasts for at least three days after treatment.These results indicated and CFA + propofol groups (n = eight neurons in four mice for each group).N) Representative traces of the action potentials recorded from PVN CRH neurons with 150 pA current injection from sham, SNI + saline, and SNI + propofol groups.Scale bars, 50 mV, 100 ms.O) Relationship between current injection and the number of elicited action potential spikes in sham, SNI + saline, and SNI + propofol groups (n = eight neurons in four mice for each group).Data are shown as the mean (white circles) ± SD (vertical lines) along with individual data points and were compared using two-tailed, unpaired Student's t-test (D, F, and K) or one-way ANOVA followed by Tukey's multiple comparisons test (H) or two-way ANOVA followed by Tukey's multiple comparisons test (M and O).CFA, complete Freund's adjuvant; SNI, spared nerve injury; PVN, paraventricular nucleus; CRH, corticotropin-releasing hormone; CFA + Saline, CFA-injected mice exposed to saline; CFA + Propofol, CFA-injected mice exposed to propofol; SNI + Saline, SNI mice exposed to saline; SNI + Propofol, SNI mice exposed to propofol.that propofol may be an effective pharmacological intervention for the treatment of pain-associated anxiety.
However, we observed that anxiety levels in mice treated with propofol did not fully return to normal.This could potentially be attributed to the administration method used in our exper-iment, which involved intraperitoneal injections.Although we took steps to enhance the mice's adaptability through handling and intraperitoneal injection training for 5 days before the experiment, it is important to note that the injection procedure itself is invasive and can serve as a stressor, potentially elevating anxiety    in mice.Furthermore, pain-associated anxiety-like behaviors are mediated by multiple brain nuclei and neural circuits, [17] including the PVN, the lateral septum (LS), the lateral hypothalamus (LH), the anterior cingulate cortex (ACC), the medial prefrontal cortex (mPFC), the basolateral amygdala (BLA), and the ventral hippocampus (vHPC).Propofol may exert its effects on certain nuclei, whereas others may not respond to its anxiolytic properties.It is worth noting that the saline plantar-injected mice used for comparison in our behavioral tests were from a different batch than the propofol-treated mice.Mice from different batches exhibited slight variations in their baseline behavioral activities, which could contribute to the differences in anxiety levels observed between the groups.

PVN CRH Neurons Serve as a Neural Target for the Anxiolytic Effects of Propofol
PVN CRH neurons play a crucial role in integrating and regulating stress and stress-induced negative emotions. [18]In response to pain, PVN CRH neurons initiate the pain response via the HPA axis neuroendocrine pathway, which ultimately leads to anxietylike behaviors. [19]4a,20] Thus, inhibiting the excessive activation of PVN CRH neurons during pain conditions may be an effective treatment for reducing pain-induced anxiety-like behaviors.Indeed, our results indicate that propofol alleviates anxiety-like behaviors associated with pain by inhibiting PVN CRH neurons.Furthermore, our results revealed that local administration of propofol to the PVN significantly mitigated pain-associated anxiety-like behaviors.These findings support our hypothesis that PVN is a critical target for the anxiolytic effects of propofol.Given that local administration circumvents the systemic circulation and directly influences the neural substrates implicated in anxiety, this approach provides a more precise demonstration of the mechanistic action of propofol on anxiety regulation.
Our study indicates that propofol-induced modifications in the activity of PVN CRH neurons primarily affect mood alterations associated with pain without exerting a direct effect on pain perception.This finding is in line with a clinical report suggesting that propofol does not improve postoperative pain outcomes in patients. [21]iven the complexity of the neuro-pathological mechanisms underlying pain-induced anxiety-like behaviors, it is important to note that the PVN CRH neurons are not the sole contributors.11c,22] Although these neuronal nuclei and circuits play a collective role in modulating anxietylike behaviors and pain thresholds, it is important to consider the possibility of their synergistic involvement in the propofolinduced alleviation of pain-associated anxiety-like behaviors.Future research should focus on investigating the specific role of these additional neuronal nuclei in the anxiolytic effects of propofol.

GABA A 𝜷3 Subunits Serve as Molecular Targets for the Anxiolytic Effects of Propofol
At the cellular level, an imbalance between excitatory and inhibitory signals may result in changes in neuronal excitability, which may contribute to negative emotions. [23]In our study, we observed a notable decrease in the frequency and amplitude of sIPSCs and an increase in the frequency of sEPSCs in PVN CRH neurons in mice exhibiting pain-induced anxiety-like behaviors compared to the control mice, which may have resulted from the disruption of the E-I balance that ultimately led to the hyperactivation of PVN CRH neurons.Propofol administration rebalanced the E-I balance by increasing sIPSCs rather than decreasing sEPSCs to reduce the hyperactivity of PVN CRH neurons.This result is consistent with previous reports indicating that clinical concentrations of propofol do not significantly affect excitatory transmission. [24]We observed similar results in the chronic pain model.The frequency and amplitude of sIPSCs in PVN CRH neurons were decreased in the SNI mice, whereas propofol restored these reductions.These findings are consistent with those of previous studies that have reported that the balance of excitatory and inhibitory inputs in PVN CRH neurons plays an essential role in regulating anxiety disorders. [8,25]The amplitude of sIPSCs is strongly linked to the expression and function of postsynaptic GABA receptors.Propofol acts as a GABA A agonist and directly activates GABA A to potentiate its response to GABA. [26]Therefore, by acting on postsynaptic GABA receptors, propofol may re-balance the E-I balance to alleviate anxiety-like behaviors associated with pain.
Modern transcriptomic and proteomic analyses have suggested that GABA A  and GABA A  subunits are the main receptor targets of propofol. [27]The  subunit of GABA A plays a role in regulating pain sensation. [28]As 100 mg kg −1 propofol did not have analgesic effects, we suggest that the  subunit may not be the main target of propofol's anxiety-relieving effects.The GABA A 3 subunit is one of the most important center zone during the OF test in scramble + propofol and Gabrb3 + propofol groups in CFA mice at 4 h after treatment (n = seven mice per group).G) Total distance traveled during the OF test in scramble + propofol and Gabrb3 + propofol groups in CFA mice at 4 h after treatment (n = seven mice per group).H) Schematic of virus injection to express Gabrb3 (GABA A 3-specific) or scramble shRNA on PVN CRH neurons from SNI mice.I) Successful expression of Gabrb3 (GABA A 3-specific) shRNA on PVN CRH neurons from SNI mice.Scale bar, 200 μm.J) Time in the open arms during the EPM test for scramble + propofol and Gabrb3 + propofol groups in SNI mice at 4 h after treatment (n = seven mice per group).K) Time in the center zone during the OF test in scramble + propofol and Gabrb3 + propofol groups in SNI mice at 4 h after treatment (n = seven mice per group).L) Total distance traveled during the OF test in scramble + propofol and Gabrb3 + propofol groups in SNI mice at 4 h after treatment (n = seven mice per group).Data are shown as the mean (white circles) ± SD (vertical lines) along with individual data points and were compared using one-way ANOVA followed by Tukey's multiple comparisons test (B) or two-tailed, unpaired Student's t-test (E-G and J-L).CFA, complete Freund's adjuvant; SNI, spared nerve injury; PVN, paraventricular nucleus; CRH, corticotropin-releasing hormone; EPM, elevated plus maze; OF, open field; Gabrb3, GABA A 3, -aminobutyric acid type A receptor 3 subunits; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.A schematic summary for how propofol alleviates anxiety-like behaviors associated with pain by inhibiting PVN CRH neurons via GABA A 3 subunits.A single dose of propofol (100 mg kg −1 ) alleviates anxiety-like behaviors associated with pain.This therapeutic effect is achieved by acting on GABA A 3 subunits, thereby restoring E-I balance and subsequently inhibiting the excessive activation of PVN CRH neurons in pain mice.CFA, complete Freund's adjuvant; SNI, spared nerve injury; PVN, paraventricular nucleus; CRH, corticotropin-releasing hormone; GABA A 3, -aminobutyric acid types A receptor 3 subunit; sIPSCs, spontaneous inhibitory postsynaptic currents.27b] The 3 subunit is highly expressed in PVN CRH neurons, [16] and has a robust effect on anxiety-related behaviors. [15,29]These previous reports are consistent with our finding that propofol reversed the decrease in GABA A 3 subunit expression in the PVN caused by the CFA injection.Indeed, our targeted downregulation of the 3 subunits in PVN CRH neurons through RNA interference prevented the anxiety-relieving properties of a single dose of propofol.However, downregulation of the 3 subunit in PVN CRH neurons did not affect the threshold of mechanical hyperalgesia in naïve mice.Based on our findings, it is reasonable to conclude that the GABA A 3 subunit may be a molecular target for propofol's anxiety-relieving effects, although we cannot rule out the role of other subunits of the GABA A in propofol's anxiety-relieving effects.28d] Defining the precise molecular mechanism through which propofol regulates the activity of PVN CRH neurons by GABA A 3 subunits to alleviate anxiety-like behaviors will require further investigation.
This study has several limitations.First, all behavioral experiments were conducted using male mice, and we did not assess the effects of propofol on anxiety-like behaviors in female mice.As sex hormone levels are closely related to anxiety, [30] the anxiety-relieving effects of propofol may differ between the sexes.Second, we selected the EPM and OF tests as indicators of anxiety-like behaviors.More behavioral paradigms, such as the light/dark box test, should be explored in future studies.
In conclusion, our findings indicate that a single dose of propofol (100 mg kg −1 ) can effectively rebalance E-I balance by acting on GABA A 3 subunits to suppress the hyperactivation of PVN CRH neurons, ultimately alleviating anxiety-like behaviors associated with pain.Our results suggest that propofol can be administered to treat other psychiatric and emotional disorders.

Experimental Section
Animals: Adult male C57BL/6J mice (8-12 weeks old) were purchased from Shanghai Model Organisms and used to establish an inflammatory pain model and a chronic pain model for behavioral tests.CRH-ires-Cre (CRH-Cre) knock-in male mice (strain no.012704) (Jackson Laboratory, Bar Harbor, ME, USA) were used for behavioral testing, fiber photometry recording and in vitro brain slice experiments.The cages were housed four or five mice per cage under a 12 h light/dark cycle (lights on from 7 am to 7 pm) at 22-25 °C with food and water ad libitum.All experiments were approved by the Institutional Animal Care and Use Committee of Shang-haiTech University (20221028002).
Drugs and Inflammatory Pain Model: An inflammatory pain model was first established.CFA (25 μL; Sigma-Aldrich, St. Louis, MO, USA) was injected into the left hind paw of each mouse using an insulin syringe to induce persistent inflammatory pain. [11,31]The control group was injected with an equal volume of saline solution.Mice had 25, 50, or 100 mg kg −1 of propofol (AstraZeneca UK Limited, London, UK) injected intraperitoneally or were injected with a similar volume of intralipid (Haisco Pharmaceutical Group, Chengdu, China) to exclude the effects of the solvent.
Spared Nerve Injury (SNI) Model: Surgery was performed under isoflurane anesthesia.The skin and muscles of the left thigh were incised to expose the sural, common peroneal, and tibial nerves.Subsequently, the nerves were explored.After exploration, non-absorbent 6-0 chromic gut sutures were used to ligate the common peroneal and tibial nerves, which were transected, and ≈2 mm sections from the dot were removed.For the sham mice, the procedure was the same as that for the experimental group, except that the nerves were left intact. [32]Twenty-one days after the SNI surgery, the mice received an intraperitoneal injection of propofol (100 mg kg −1 ).
Behavioral Assays: Behavioral tests were performed when mice were 8-12 weeks old.32a] Behavioral tests were performed at 4 h; 24 h; or 3 days after propofol treatment.To eliminate the impact of possible adaptation on performance, each mouse underwent behavioral tests only once, and separate cohorts were used for the different time points.
Elevated Plus Maze (EPM) Test: The EPM apparatus consisted of two closed arms (35 cm × 6 cm) and two open arms (35 cm × 6 cm), with a central platform (6 cm × 6 cm) at the confluence.Each mouse was placed on the central platform facing an open arm and was allowed to explore the maze for 5 min.11c] Open Field (OF) Test: The OF apparatus consisted of a central square area (25 cm × 25 cm), with surrounding margins (50 cm × 50 cm × 60 cm).11c] The total distance traveled and time spent in the central zone were calculated using behavioral software (Jiliang Software, Shanghai, China).
Mechanical Sensitivity: The mechanical withdrawal thresholds were measured using von Frey filaments.Mice were placed individually in a plastic chamber and allowed to acclimate for 1 h, after which they were tested for the paw withdrawal thresholds.von Frey filaments were used in ascending order to exert 0.02 to 1.4 g in the middle of the left hind paw surface.11c] Enzyme-Linked Immunosorbent Assay (ELISA): Blood samples were collected from the experimental mice (the sampling time points were consistent with the behavioral testing time points) and centrifuged at 15 g for 15 min.The resulting serum samples were promptly frozen at −80 °C until further use.Serum CORT, CRH, and NE levels were measured using ELISA Kits (Jiangsu Jingmei Biological Technology Co., Ltd., Yancheng, China).
Fiber Photometry Recording: The optical fiber (outer diameter, 200 μm; numerical aperture, 0.37; Anilab) was inserted into the ceramic ferrule and introduced into the PVN after the rAAV-hSyn-DIO-GCaMP7b virus injection.Fluorescent signals were acquired using a fiber photometry system (including a 488 nm excitation laser, 505-to 544-nm emission filter and a photomultiplier tube [R3896]; Hamamatsu).The signals were recorded using Spike 2 software (CED, Cambridge, UK).Photometry data were exported from Spike 2 to MATLAB R2020b Mat files for analysis.The values of fluorescence changes (ΔF/F) were derived by calculating (F−F 0 )/F 0 , where F 0 was the baseline fluorescence signal averaged over a 5-s-long control time window.The ΔF/F values were presented as average plots to illustrate the signal changes trial by trial.The analytical procedures were similar to those described previously. [8]hemogenetic Inhibition of PVN CRH Neurons: PVN CRH neurons were bilaterally injected with rAAV-hSyn-DIO-hM4D(Gi)-mCherry or rAAV-hSyn-DIO-mCherry.The mice were placed in the testing room for 1 h before the behavioral tests.Behavioral tests were performed 1 h after CNO (3 mg kg −1 , i.p.) was injected into each mouse.
Chemogenetic Activation of PVN CRH Neurons: PVN CRH neurons were bilaterally injected with rAAV-hSyn-DIO-hM3D(Gq)-mCherry or rAAV-hSyn-DIO-mCherry.The mice were placed in the testing room 1 h before the behavioral tests to allow acclimation.CNO (3 mg kg −1 , i.p.) was injected into each mouse in the treated group 30 min before propofol injection, whereas an equal volume of saline was injected into each mouse in the control group.Behavioral tests were performed 4 h after the propofol or saline injection.
Cannula Infusion: A double guide cannula (center-to-center distance 0.3 mm, RWD) was placed above the PVN.After the mice had recovered for at least 14 days, propofol (10 μm) was microinjected with a double injector cannula, which had a 0.5-mm extension beyond the tip of the guide cannula.
4a] Adult (6-8 weeks old) male CRH-ires-Cre mice were injected with rAAV-hSyn-DIO-mCherry into the PVN.To induce acute pain: two weeks later, mice were injected with CFA in their left hind paws and injected intraperitoneally with propofol or saline.To induce chronic pain: the SNI surgery was performed on the mice after virus injection.Twenty-one days later, the SNI mice were injected with saline or propofol (100 mg kg −1 ).Four hours after treatment, the mice were anaesthetized with tribromoethanol and perfused with ice-cold oxygenated (95% O2; 5% CO2) NMDG artificial cerebrospinal fluid (ACSF) solution, which consisted of 93 mm NMDG, 93 mm HCl, 2.5 mm KCl, 1.25 mm NaH2PO4, 10 mm MgSO4•7H2O, 30 mm NaHCO3, 25 mm glucose, 20 mm HEPES, 5 mm sodium ascorbate, 3 mm sodium pyruvate, and 2 mm thiourea.After perfusion, the brain was rapidly dissected and immediately transferred to an ice-cold oxygenated NMDG ACSF solution.Brain tissue was sectioned in the coronal plane at a thickness of 300 mm using a vibratome (VT1200 S, Leica, Wetzlar, Germany).Brain slices were incubated in oxygenated NMDG ACSF at 32 °C for 10-15 min and then transferred into normal oxygenated ACSF (126 mm NaCl, 2.5 mm KCl, 1.25 mm NaH2PO4, 2 mm MgSO4•7H2O, 10 mm glucose, 26 mm NaHCO3, and 2 mm CaCl2) at room temperature for 1 h.All chemicals used in slice preparation were purchased from Sigma-Aldrich.
Slices were transferred to the recording chamber with ACSF perfusion at a rate of 3 mL min −1 at 28 °C.Whole-cell patch-clamp recordings were made from PVN CRH neurons and visualized with an Olympus BX61W1 microscope (equipped with GFP and mCherry filters) using infrared video microscopy and differential interference contrast optics.Recording pipettes (3-4 MΩ) were prepared using a micropipette puller (P2000, Sutter Instrument, USA).For whole-cell recording, the pipettes were filled with the ACSF solution containing 133 mm potassium gluconate, 18 mm NaCl, 0.6 mm EGTA, 10 mm HEPES, 2 mm MgATP, and 0.3 mm Na3•GTP (pH, 7.2; 280 mOsm).For APs evoked by current injections, a current-step protocol (from −25 to 200 pA, with 25-pA increments for PVN recording) was run and repeated.The neurons were held at −70 mV in voltage-clamp mode to record sIPSCs and sEPSCs for 5 min.
Western Blot: Total proteins were extracted from the PVN to detect the expression of GABA A 3 subunits.RIPA buffer working solution was used to extract proteins.The proteins in each sample were quantified using a bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, MA, USA).Equal amounts of protein (20 μg) were electrophoresed on a 10% SDS-polyacrylamide gel followed by electrotransfer onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA) (Figure S15, Supporting Information).
Membranes were blocked for 2 h in TBST (150 mm NaCl, 10 mm Tris, 0.1% Tween 20, pH 7.6) containing 10% BSA.The primary antibody against GABA A 3 (Abcam, Cambridge, UK, ab300063-40μL) was diluted in blocking buffer and incubated overnight at 4 °C.The blots were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature after three washes in phosphate-buffered saline (PBS).The blots were exposed to an enhanced chemiluminescent substrate after three additional washes in PBS.Densitometric analysis of protein bands was performed by an independent blinded observer using Image J software.
4a,34] In our experiments, three mice were excluded from data collection and analysis because of failed fiber implantation, and nine mice were excluded from data collection and analysis because of virus injection misalignment.Data were analyzed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) or MATLAB R2020b.Unpaired Student's t-test was used for comparisons between two independent groups.Data were subjected to one-way ANOVA followed by Tukey's multiple comparisons test if they passed the Kolmogorov-Smirnov normality test or followed by the Kruskal-Wallis's multiple comparisons test if they failed the normality test. [35]Two-way ANOVA followed by Tukey's comparison test was used for multiple comparisons. [36]Data were presented in the text as the mean and standard deviation (SD) in the form "mean ± SD".P<0.05 was considered statistically significant.

Figure 1 .
Figure 1.Propofol alleviates anxiety-like behaviors and the stress-related hormones surge associated with pain.A) Schematic of the injection of CFA and propofol along with behavioral and stress-related hormones tests.B) Paw withdrawal threshold over time for saline and CFA groups (n = eight mice per group).C) Time in the open arms during the EPM test for saline and CFA groups at 4 h after saline or CFA injection (n = eight mice per group).D) Time in the central zone during the OF test for saline and CFA groups at 4 h after saline or CFA injection (n = eight mice per group).E) Total distance traveled during the OF test for saline and CFA groups at 4 h after saline or CFA injection (n = eight mice per group).F) Paw withdrawal threshold over time for CFA + saline and CFA + propofol (100 mg kg −1 ) groups at 4 h after drug injection (n = eight mice per group).G) Time in the open arms during the

Figure 2 .
Figure 2. The response dynamics of PVN CRH neurons to pain and propofol.A) Schematic of fiber implantation above PVN CRH neurons expressing GCaMP7b in CRH-ires-Cre mice.B) Successful expression of GCaMP7b in PVN CRH neurons.Scale bar, 200 μm.C) Calcium signaling as determined by GCaMP7b fluorescence in PVN CRH neurons relative to the moment of intraplantar injection of saline or CFA (n = seven mice per group).D) Quantification of changes in GCaMP7b signals after administration of saline and CFA.E) Calcium signaling as determined by GCaMP7b fluorescence in PVN CRH neurons relative to the moment of administration of saline or propofol (100 mg kg −1 ) (n = six mice per group).F) Quantification of changes in GCaMP7b signals after administration of saline and propofol (100 mg kg −1 ).G) GCaMP7b signals from PVN CRH neurons over three periods: the baseline value before treatment, after CFA injection, and after propofol or saline injection (n = seven mice per group).H) Quantification of changes in GCaMP7b signals during these three periods.I) Schematic of fiber implantation above PVN CRH neurons expressing GCaMP7b in CRH-ires-Cre mice.J) Calcium signaling as determined by GCaMP7b fluorescence in PVN CRH neurons relative to the moment of administration of saline or propofol in SNI mice (100 mg kg −1 ) (n = six mice per group).K) Quantification of changes in GCaMP7b signals after administration of saline and propofol (100 mg kg −1 ) in SNI mice.L) Representative traces of the action potentials recorded from PVN CRH neurons with 125 pA current injection from control, CFA + saline, and CFA+propofol groups.Scale bars, 75 mV, 150 ms.M) Relationship between current injection and the number of elicited action potential spikes in control, CFA + saline,

Figure 3 .
Figure 3. Inhibiting PVN CRH neurons alleviates anxiety-like behaviors associated with pain.A) Schematic of viral injection (mCherry or hM4D(Gi)) along with behavioral tests in CFA mice.B) Time in the open arms during the EPM test for mCherry and hM4D(Gi) groups in CFA mice (n = seven mice per group).C) Time in the central zone during the OF test for mCherry and hM4D(Gi) groups in CFA mice (n = seven mice per group).D) Total distance traveled during the OF test for mCherry and hM4D(Gi) groups in CFA mice (n = seven mice per group).(E) Paw withdrawal threshold for mCherry and hM4D(Gi) groups in CFA mice (n = seven mice per group).F) Schematic of viral injection (mCherry or hM4D(Gi)) along with behavioral tests in SNI mice.G) Time in the open arms during the EPM test for mCherry and hM4D(Gi) groups in SNI mice (n = seven mice per group).H) Time in the central zone during the OF test for mCherry and hM4D(Gi) groups in SNI mice (n = seven mice per group).I) Total distance traveled during the OF test for mCherry and hM4D(Gi) groups in SNI mice (n = seven mice per group).J) Paw withdrawal threshold for mCherry and hM4D(Gi) groups in SNI mice (n = seven mice per group).Data are shown as the mean (white circles) ± SD (vertical lines) along with individual data points and were compared using two-tailed, unpaired Student's t-test (B-D and G-I) or two-way ANOVA followed by Tukey's multiple comparisons test (E and J).CFA, complete Freund's adjuvant; SNI, spared nerve injury; EPM, elevated plus maze; OF, open field; PVN, paraventricular nucleus; CRH, corticotropin-releasing hormone; BL, baseline; CNO, Clozapine N-oxide.

Figure 5 .
Figure 5. Propofol rebalances the synaptic homeostasis of PVN CRH neurons by increasing inhibition.A) Schematic of whole-cell patch-clamp recording in the PVN brain slice from CFA mice.B) Representative traces showing sIPSCs in PVN CRH neurons from an individual mouse for each group.Scale bars, 50 pA, 2.5 s.C) Average frequency of sIPSCs in different groups at 4 h after treatment (control, n = nine neurons in four mice; CFA + saline, n = seven neurons in four mice; CFA + propofol, n = nine neurons in four mice).D) Average amplitude of sIPSCs in different groups at 4 h after treatment (control, n = nine neurons in four mice; CFA + saline, n = seven neurons in four mice; CFA + propofol, n = nine neurons in four mice).E) Representative traces showing sEPSCs in PVN CRH neurons from an individual mouse for each group.Scale bars, 40 pA, 2 s.F) Average frequency of sEPSCs in different groups at 4 h after treatment (control, n = nine neurons in four mice; CFA + saline, n = ten neurons in four mice; CFA + propofol, n = eight neurons in four mice).G) Average amplitude of sEPSCs in different groups at 4 h after treatment (control, n = nine neurons in four mice; CFA + saline, n = ten neurons in four mice; CFA + propofol, n = eight neurons in four mice).H) Schematic of whole-cell patch-clamp recording in the PVN brain slice from SNI mice.I) Representative traces showing sIPSCs in PVN CRH neurons from an individual mouse for each group.Scale bars, 50 pA, 5 s.J) Average frequency of sIPSCs in different groups at 4 h after treatment (sham, n = ten neurons in four mice; SNI + saline, n = ten neurons in four mice; SNI + propofol, n = ten neurons in four mice).K) Average amplitude of sIPSCs in different groups at 4 h after treatment (sham, n = ten neurons in four mice; SNI + saline, n = ten neurons in four mice; SNI + propofol, n = ten neurons in four mice).L) Representative traces showing sEPSCs in PVN CRH neurons from an individual mouse for each group.Scale bars, 50 pA, 5 s.M) Average frequency of sEPSCs in different groups at 4 h after treatment (sham, n = ten neurons in four mice; SNI + saline, n = ten neurons in four mice; SNI + propofol, n = ten neurons in four mice).N) Average amplitude of sEPSCs in different groups at 4 h after treatment (sham, n = ten neurons in four mice; SNI + saline, n = ten neurons in four mice; SNI + propofol, n = ten neurons in four mice).Data are shown as the mean (white circles) ± SD (vertical lines) along with individual data points and were compared using one-way ANOVA followed by Tukey's multiple comparisons test (C, D, F, J, and N) or one-way ANOVA followed by Kruskal-Wallis's multiple comparisons test (G, K, and M).CFA, complete Freund's adjuvant; SNI, spared nerve injury; PVN, paraventricular nucleus; CRH, corticotropin-releasing hormone; CFA + Saline, CFA-injected mice exposed to saline; CFA + Propofol, CFA-injected mice exposed to propofol; SNI + Saline, SNI mice exposed to saline; SNI + Propofol, SNI mice exposed to propofol; sIPSCs, spontaneous inhibitory postsynaptic currents; sEPSCs, spontaneous excitatory postsynaptic currents.

Figure 6 .
Figure 6.Propofol effects on and dependence on GABA A 3 subunits in PVN CRH neurons.A) Representative image of GABA A 3 immunoblot with saline + saline, saline + propofol, CFA + saline, and CFA + propofol samples.Proteins were isolated from the PVN tissue and GAPDH served as a loading control.B) Relative expression level of GABA A 3 in the PVN tissue from saline + saline, saline + propofol, CFA + saline, and CFA + propofol mice (n = four mice per group).C) Schematic of virus injection to express Gabrb3 (GABA A 3-specific) or scramble shRNA on PVN CRH neurons from CFA mice.D) Successful expression of Gabrb3 (GABA A 3-specific) shRNA on PVN CRH neurons from CFA mice.Scale bar, 200 μm.E) Time in the open arms during the EPM test for scramble + propofol and Gabrb3 + propofol groups in CFA mice at 4 h after treatment (n = seven mice per group).F) Time in the levels

Figure 7 .
Figure 7.A schematic summary for how propofol alleviates anxiety-like behaviors associated with pain by inhibiting PVN CRH neurons via GABA A 3 subunits.A single dose of propofol (100 mg kg −1 ) alleviates anxiety-like behaviors associated with pain.This therapeutic effect is achieved by acting on GABA A 3 subunits, thereby restoring E-I balance and subsequently inhibiting the excessive activation of PVN CRH neurons in pain mice.CFA, complete Freund's adjuvant; SNI, spared nerve injury; PVN, paraventricular nucleus; CRH, corticotropin-releasing hormone; GABA A 3, -aminobutyric acid types A receptor 3 subunit; sIPSCs, spontaneous inhibitory postsynaptic currents.